Novel compounds

ABSTRACT

An antibody, or a derivate or a fragment thereof, having a binding structure for a target structure is described. The antibody is displayed in, and on the cell surface of, human gastrointestinal epithelial tumour cells and in a subpopulation of normal human gastrointestinal epithelial cells. Said binding structure comprises the complementarity determining region (CDR) sequences in the light chain comprising essentially the amino acids number 23-33 (CDR1), 49-55 (CDR2), 88-98 (CDR3) of the amino acid sequence shown in SEQ ID NO:2, and the CDR sequences in the heavy chain comprising essentially the amino acids number 158-162 (CDR1), 177-193 (CDR2, 226-238 (CDR3) of the amino acid sequence shown in SEQ ID NO:2, or other binding structures with similar unique binding properties. There is also described a target structure displayed in, or on the surface of tumour cells, vaccine compositions, pharmaceutical compositions as well as methods related to human malignant diseases.

The present invention is related to an antibody, or a derivate, or afragment thereof, having a binding structure for a target structuredisplayed in, and on the cell surface of, human gastrointestinalepithelial tumour cells and in a subpopulation of normal humangastrointestinal epithelial cells; and to a target structure displayedin, or on the surface of tumour cells; vaccine compositions;pharmaceutical compositions; as well as methods related to humanmalignant diseases.

BACKGROUND OF THE INVENTION

Surgery is the primary treatment of colorectal cancer leading tofive-year survival rates of 90 to 40 percent depending on the state oftumour progression from Dukes Stage A to C. Conventional adjuvanttherapy that includes radiation therapy and chemotherapy has been ableto reduce the death rates further by approximately 30 percent (1).Despite these achievements cancer of the colon and rectum is one of themajor causes of death in human cancer. Immunological therapy has beenextensively attempted. However, colon cancer has generally beenresistant to immunotherapy and is considered to be of lowimmunogenicity. Patients with colon cancer neither respond to IL-2treatment or adoptive transfer of in vitro cultured tumour infiltratinglymphocytes otherwise active in patients with immunogenic malignanciessuch as melanoma. Most encouraging however, Riethmüller et al. reporteda 32 percent decreased seven-year death rate for Dukes Stage Ccolorectal cancer treated after primary tumour resection with a nakedmurine mAb directed to a tumour and normal epithelial associated antigen(Ep-CAM) (2), indicating that other immunotherapeutic modalities couldbe effective.

A significant improvement of adjuvant immunotherapy and of the treatmentof more advanced stages of cancer should require a more potent effectormechanism than provided by a naked mAb. In principle, an increasedpotency should require an increased tumour selectivity of the targetingantibody.

The limited number of colon cancer associated antigens defined todayhave been discovered using hybridoma produced murine mAbs resulting fromxenogenic immunisations with human tumours (3).

The use of large phage display libraries for the identification of noveltumour-associated antigens can be expected to significantly speed up theprocess of finding target molecules useful for tumour immunotherapy anddiagnosis. Such identification of target molecules could be accomplishedby the selection and screening of antibody phage libraries on culturedtumour cells and tissue sections to generate specific reagents definingin vitro and in vivo expressed antigens (4). The phage displaytechnology has been established as an efficient tool to generatemonoclonal antibody reagents to various purified antigens, and theconstruction and successful selection outcome from immune, naive andsynthetic antibody phage libraries have been described in severalstudies (5).

Non-immune libraries are favourable with respect to their generalapplicability, making unique libraries for every single targetunnecessary. On the other hand, sufficiently large and high qualitynon-immune libraries are difficult to construct and a target discoveryprocess using these libraries should require efficient subtractiveselection methods when based on complex antigens.

A phage library of a more moderate size has now been constructed from anear human primate immunised with complex human antigens. Thisrepresents an approach that takes advantage of an in vivo pre-selectedrepertoire. Such libraries should be enriched for specificities totumour specific epitopes in a reduced background reactivity toxenogeneic antigens (6). Furthermore, as compared to the mouse, primateantibodies demonstrating close sequence homology with human antibodiesshould not be immunogenic in man (7).

Novel primate antibodies from a phage library that define selectivelyexpressed colon cancer associated antigens have now been identified. Thetherapeutic potential, demonstrated by T cell mediated killing ofcultured colon cancer cells coated with two of these antibodies fused toengineered superantigens, is comparable with superantigens fused tomurine Fab fragment specific for colon cancer associated antigens suchas EP-CAM, for which there has previously been established thetherapeutic capacity in experimental systems (8).

There is also provided a method for efficient positive and subtractivecell selection of phage antibodies that should facilitate futureidentification of novel phenotype specific antigens including tumourassociated antigens using antibodies from large phage libraries.

BRIEF SUMMARY OF THE INVENTION

The present invention is related in a first aspect to an antibody, or aderivative or a fragment thereof, having a binding structure for atarget structure displayed in, and on the cell surface of, humangastrointestinal epithelial tumour cells and in a subpopulation ofnormal human gastrointestinal epithelial cells, said binding structurecomprising the complementarity determining region (CDR) sequences in thelight chain comprising essentially the amino acids number 23-33 (CDR1),49-55 (CDR2), 88-98 (CDR3) of the amino acid sequence shown in SEQ IDNO:2, and the CDR sequences in the heavy chain comprising essentiallythe amino acids number 158-162 (CDR1), 177-193 (CDR2), 226-238 (CDR3) ofthe amino acid sequence shown in NO: 2, or other binding structures withsimilar unique binding properties.

In one embodiment the antibody is phage selected. In another embodimentthe sequences are of Macaca fascicularis origin. A further embodiment ofthe invention is a derivative of said antibody, which derivative is ofhuman origin. The sequences preferably have an identity of at least 84%to corresponding sequences of human origin. Preferably, the antibody haslow immunogenicity or non-immunogenicity in humans.

In a further embodiment, the antibody has been derivatised bygenetically linking to other polypeptides, and/or by chemicalconjugation to organic or non-organic chemical molecules, and/or by di-,oligo- or multimerisation.

In still a further embodiment, said antibody is genetically linked orchemically conjugated to cytotoxic polypeptides or to cytotoxic organicor non-organic chemical molecules.

In a further embodiment, said antibody is genetically linked orchemically conjugated to biologically active molecules.

In still a further embodiment, said antibody is genetically linked orchemically conjugated to immune activating molecules.

In another embodiment, said antibody has been changed to increase ordecrease the avidity and/or affinity thereof.

In still another embodiment, said antibody has been changed to increasethe production yield thereof.

In a further embodiement, said antibody has been changed to influencethe pharmacokinetic properties thereof.

In still a further embodiment, said antibody has been changed to givenew pharmacokinetic properties thereto.

In a further embodiment, said antibody is labeled and the bindingthereof is inhibited by an unlabeled form of said antibody and not byother binding structures, and not inhibiting the binding of otherbinding structures having other specificities.

A further embodiment is an antibody, the binding structure of whichrecognizes a non-reduced form of α6β4 integrin.

In another aspect the invention relates to a target structure displayedin, or on the surface of, tumour cells, said target structure

a) having the ability of being specifically blocked by and tospecifically block the binding structure of an antibody as defined inany one of claims 1-14, and other binding structures with similarbinding specificities,

b) being displayed in, and on the surface of, human gastrointestinalepithelial cells,

c) having substantial homology with α6 and/or β4 integrin chains orvariants thereof, representing a shared or unique epitope,

d) being highly expressed on the surface of tumour cells, and

e) being a target for cytotoxic effector mechanisms.

By substantial homology in this context is meant homology in those partsof the target structure which are relevant for the binding of theantibody.

In one embodiment of said target structure, the binding structure islabeled and the binding thereof is inhibited by an unlabeled form ofsaid binding structure and not by other binding structures, and notinhibiting the binding of other binding structures having other bindingspecificies.

In a further embodiment of said target structure said binding structurecomprises one or more of the complementarity determining region (CDR)sequences comprising essentially the amino acids number 23-33, 49-55,88-98, 158-162, 177-193, 226-238 of the amino acid sequence shown in SEQID NO:2, or other binding structures with similar unique bindingproperties.

In still a further embodiment of said target structure said bindingstructure is an antibody, which antibody in a further embodimentcomprises the variable region of a light chain comprising essentiallythe amino acids number 1-109 of the amino acid sequence shown in SEQ IDNO:2, and the variable region of a heavy chain comprising essentiallythe amino acids number 128-249 of the amino acid sequence shown in SEQID NO: 2.

Said target structure is in a further embodiment expressed homogenouslyin human colonic epithelial cells and less in pancreatic duct and bileduct cells.

In still a further embodiment, the expression of said target structureis correlated to gastrointestinal epithelial differentiation.

In another embodiment, said target structure comprises the amino acidsequence of α6β4 integrin, of which the α6 part is shown in SEQ ID NO: 3and the β4 part is shown in SEQ ID NO: 4. Another embodiment of thetarget structure comprises homo- or heteromonomers or homo- orheteromultimers of said α6β4 integrin and/or of said one or morefragments and/or variants and/or subunits thereof. Preferably, saidtarget structure has an apparent molecular weight in its non-reducedform of from 90 to 140 kDa, most preferred fro 80 to 160 kDa.

In still further embodiments the target structure compirses a peptide orpolypeptide(s) comprising essentially any one of the amino acidsequences shown in SEQ ID NOs: 5-51, or comprises a molecule complexedto said polypeptide(s).

In the case of a target structure comprising amino acid sequences fromthe α6β4 integrin, said target structure may in a further embodiment berecognised, exclusively or not, in its non-reduced form by the bindingstructure comprised by the antibody as defined above.

The invention relates in a further aspect to a substance which binds tothe target structure as defined above, which substance is an organicchemnical molecule or a peptide. In one embodiment, said substance is ananti-idiotype of said target structure. Said anti-idiotype may bespecifically blocked by and specifically block a binding structurehaving similar binding specificity for said target structure.

In a still further aspect, the invention relates to a substance thatblocks the function of the target structure as defined above, whichsubstance is an organic molecule or a peptide.

In another aspect, the invention relates to a binding structure whichrecognises a target structure as defined above and which is of anorganic chemical nature.

In a further aspect, the invention relates to a pharmaceuticalcompositin comprising as an active principle an antibody as definedabove, or a target structure as defined above, or a substance as definedabove.

In still a further aspect, the invention is related to a vaccinecomposition comprising as an active principle abn antibody as definedabove, or a target structure as defined above, or a substance as definedabove.

In a further aspect, the invention is related to a method of therapy fortreating conditions based on an anti-angiotenic mechanism, whereby anantibody as defined above, or a target structure as defined above, or asubstance as defined above, is administered to a human subject.

In another aspect, the invention is related to a method of treatinghuman metastatic diseases, wherein an antibody as defined above isadministered to a human subject.

In a further aspect the invention is related to a method of in vitrohistopathological diagnosis and prognosis of human malignant disease,whereby a sample is contacted with an antibody as defined above and anindicator.

Embodiments of said method comprise tumour typing, tumour screening,tumour diagnosis and prognosis, and monitoring premalignant conditions.

In still a further aspect, the invention is related to a method for invitro diagnosis and prognosis of human malignant disease, wherebyconcentrations in bodily fluids of an antigen comprising a targetstructure, as defined above, or an anti-idiotype of said targetstructure, as defined above, is assayed.

A further aspect of the invention is related to a method for in vitrodiagnosis and prognosis of human malignant disease, wherebyconcentrations in bodily fluids of an antibody as defined above isassayed.

A still further aspect of the invention is related to a method for invitro diagnosis and prognosis of human malignant disease, wherebyconcentrations in bodily fluids of a complex of a) an antigen comprisinga target structure, as defined above, or an anti-idiotype of said targetstructure, as defined above, and b) an antibody, as defined above, isassayed.

In a still further aspect, the invention is related to a method for invivo diagnosis and prognosis of human malignant disease, whereby thelocalisation of an antibody, as defined above, to tumour deposits in ahuman subject is determined. Said antibody is preferably administered tothe subject before the determination. In one embodiment said antibody isaccumulated in tumour deposits. In a further embodiment, said method isquantitative.

Another aspect of the invention is related to a method for therapy ofhuman malignant disease, whereby an antibody, as defined above, isadministered to a human subject. In one embodiment of this method saidantibody has been changed by being genetically linked to moleculesgiving the combined molecule changed pharmacokinetic properties. Inanother embodiment said antibody has been changed by being derivatised.

DETAILED DESCRIPTION OF THE INVENTION

The identification of novel tumour associated antigens (TAAs) is pivotalfor the progression in the fields of tumour immunotherapy and diagnosis.In relation to the present invention, there was first developed, basedon flow cytometric evaluation and use of a mini-library composed ofspecific antibody clones linked to different antibiotic resistancemarkers, methods for positive and subtractive selection of phageantibodies employing intact cells as the antigen source. An scFv phagelibrary (2.7×10⁷) was constructed from a primate (Macaca fascicularis)immunised with pooled human colon carcinomas. This library was selectedfor three rounds by binding to Colo205 colon adenocarcinoma cells, andproteolytic elution followed by phage amplification.

Several antibodies reactive with colon carcinomas and with restrictedreactivity with a few epithelial normal tissues were identified byimmunohistochemistry. One clone, A3 scFv, recognised an epitope that washomogeneously expressed in 11/11 of colon and 4/4 pancreatic carcinomasstudied and normal tissue expression restricted to subtypes of epitheliain the gastrointestinal tract. The A3 scFv had an apparent overallaffinity about 100-fold higher than an A3 Fab, indicating binding ofscFv homodimers. The cell surface density of the A3 epitope, calculatedon the basis of Fab binding, was exceptionally high, approaching 3million per cell.

Efficient T cell mediated killing of colon cancer cells coated with A3scFv fused to the low MHC class II binding superantigen mutantSEA(D227A) is also demonstrated. The identified A3 molecule thusrepresents a TAA with properties that suggests its use forimmuno-therapy of colon and pancreatic cancer.

Discussion

In relation to the present invention, efficient protocols for phageselection to be used for the identification of cell phenotype specificantibody fragments from large phage libraries was developed. The targetspecificities for the applications as exemplified were for colon tumourassociated antigens.

First the frequency of pIII-scFv fusion protein surface display in thephage population using the herein presented phagemid construct for phagepropagation was analysed. A higher level of C215 scFv display wasachieved as compared to previous reports. This should favour subtractiveselection efficiency, but also increases the probability of avidityselection of low affinity antibodies from libraries.

Specificity of C215 scFv phage binding to colon adenocarcinoma Colo205cells was clearly demonstrated. Bound phage could be efficiently elutedby use of the protease Genenase that specifically cleaves a targetsequence between the phage protein III and the scFv antibody leaving thecells intact after elution. This non-chemical elution method shouldequally efficiently elute phage antibodies irrespectively of theirbinding affinity and only phage bound by scFv interactions, adding tothe specificity of the process.

The enrichment achieved after three selection rounds on Colo205 cells(500 000×) using this selection protocol was similar to that reported byother investigators for selections on complex antigens.

After verifying the performance of the various methodological steps thecombined technology was applied to library selections using Colo205cells.

The library was constructed from a near human species immunised withhuman tumours. The antibody pool generated this way would potentiallyinclude affinity matured antibodies to tumour specific antigens in alimited background of xeno reactivities to widespread normal humantissue antigens (6). The antibodies identified recognised tumour andtissue differentiation antigens with restricted normal tissuedistribution. All of the selected antibodies identified as colon cancertissue reactive in the primary screening also reacted with viableColo205 cells in flow cytometry. This restriction to cell surfacespecificities should reflect the selection process and not thecomposition of the library, since a suspension of a mixture of tumourtissue components was used for the immunisation.

In a similar previous study extra- and intracellular specificities wereidentified in an anti-melanoma library produced the same way andselected using tissue sections as the antigen source (4). Tissuesections of resected human colorectal tumours and normal colon (mountedin the same well) were used for the primary screening usingimmunohistochemistry to assure the clinical relevance of the selectedspecificities, to increase the efficiency and to obtain more qualitativeinformation as compared to flow cytometric screening.

The selected antibodies could be classified into four antibodyspecificity groups, distinguished by their reactivity patterns toepithelia in different organs (see Example 1, Table 1). Among thesespecificity groups, A3 scFv identified the most tumour selectiveantigen. This A3 TAA was highly, homogeneously and frequently expressedin samples of primary and metastatic colon cancer and of pancreaticcancer. Furthermore, its cell surface expression level as determinedwith the A3 Fab fusion protein (3 millions epitopes/cell) wasexceptionally high and permissive for cell surface mediated cytotoxiceffects.

Few, if any, of the frequently expressed human tumour antigens definedare tumour specific, but are commonly related to tissue differentiationsuch as A3 and the Ep-CAM. However, upregulated expression of theseantigens in tumours should provide a basis for a therapeutically activedose window. The availability from the circulation of normal tissuecompartments expressing the antigen may also be more restricted due tolimited capillary permeability and their site of expression in the body(e.g. the exposure of the apical side of gut epithelial cells tocirculating antibodies should be very limited).

The clinical experience with the pan-epithelial Ep-CAM reactive 17-1AmAb supports the feasibility to identify an effective non-toxic antibodydose. The restricted expression in epithelia of all of the selected scFvclones in this work, indicate that these clones in principal could beevaluated as candidates for immuno-therapeutic applications analogouslyto the 17-1A, e.g. as full-length mAbs. However, a particular advantagefor the A3 TAA as compared to the Ep-CAM is the lack of expression inmost normal epithelia such as of the lung and kidney, although theexpression in the colon is similar.

The tissue distribution to subtypes of normal epithelia is supported bythe selective expression in subtypes of carcinomas originating from thegastro-intestinal tract (see Example 2, Table 2).

Several of the previously well-known colon cancer associated antigens(CEA, CA50, CA19-9, CA242, Tag-72) (3) are expressed equally or morerestrictedly in normal tissues as compared to the A3 epitope. However,in contrast to the A3 and the C215 Ep-CAM they are more heterogeneouslyexpressed in tumours.

Use of antibodies to the Ep-CAM has demonstrated good clinical resultsincluding a survival advantage for colorectal cancer patients in anadjuvant setting (2). With the objective to induce tumour responses evenin more advanced stage patients, the introduction of potent effectormolecules in conjunction with this antibody will challenge the “normaltissue resistance” seen in the treatment with the naked 17-1A mAb.Preclinically, this could be studied in model systems usingtoxin-conjugated antibodies specific to the murine version of thisantigen or animals transgenic for human colon cancer associatedantigens.

Previously, antibody immunotoxins have been successfully used to curemice in models with metastatically growing tumours expressing xeno(human) tumour antigens not expressed in mouse tissues (10). However,the TAAs used are truly tumour specific and the models do not reflectthe potential for normal tissue targeted toxicity.

In previous studies we have reported the potential of superantigens asimmunostimulatory toxins for tumour immunotherapy (8). Antibody mediatedtargeting of superantigens attracted large numbers of cytotoxic andcytokine-producing T cells to the tumour site. The superantigenSEA(D227A), mutated for low MHC class II binding affinity, wasgenetically linked to tumour targeting antibodies. This“tumour-selective” agent was applied to recruit T cells independent ofMHC expression in the tumour, thus short-cutting the problems of MHCdown regulation and polymorphism that represent significant obstaclesfor other active immunotherapeutic approaches.

The mini-library of the established “tumour-selective”, 1F scFv phage,the “broadly-reactive” C215 phage and the non-specific D1.3 phageantibody clones was an essential tool for the development of protocolsfor efficient subtractive cell selection. A requirement for thisselection principle is that the negative selection is followed bypositive selection before phage rescue and amplification, due to thehigh frequency of non-displaying phage particles. Alternatively,non-displaying phage can be made non-infective by selective proteolysis(G. Winter, pers. comm.). Such a technique may allow the generation of“inert libraries”, i.e. libraries that have been extensively negativelypreselected (e.g. towards a cell in a resting state or a transfectableparental cell).

In conclusion, the “non-wanted” model phage specificity couldselectively be subtracted from the phage population by a factor ofapprox. 100 for each selection round. Future subtractive selectionsusing the developed protocol in combination with the use of largenon-immune phage libraries for identification of differentiallyexpressed cell surface antigens will demonstrate whether such anapproach prove to be superior to the strategy we used in this study,i.e. positive selection using an in vivo pre-selected immune repertoire,including restrictions and biases such as immunodominance (4). The lowaffinity and high epitope density demonstrated for the A3 Fab binding totumour cells as compared to the A3 scFv fusion protein suggestsformation of scFv multimers that interact with epitopes that cluster oncell surfaces. Higher affinity monovalent variants of A3 Fab oralternatively, stable divalent constructs such as full-length A3 Fvgrafted mAbs compatible with the putative low immunogenicity of A3should be developed. Such constructs would be suitable for targeting ofappropriate effector molecules to this highly expressedgastro-intestinal tumour associated antigen.

The invention is further illustrated in the following nonlimitingexperimental part of the description.

EXMPERIMENTAL PART

Materials and Methods

Animals

Cynomolgus Macaque (Macaca fascicularis) monkeys were kept and immunisedat the Swedish Institute for Infectious Decease Control (SIIDC),Stockholm. Water and food were always available ad libitum. Four monkeyswere immunised subcutaneously with 2 ml of a crude suspension of coloncancer tissues in 10% normal cynomolgus serum in PBS. Booster doses weregiven day 21, 35, and 49. Antibody responses were demonstrated in twomonkeys where the antigen had been admixed with alum adjuvant. Allanimals were kept according to Swedish legislation and the experimentswere approved by the local ethical committees.

Tissues and Cells

Human tumours and normal tissue samples were obtained from LundUniversity Hospital and Malm{umlaut over (o )} General Hospital, Sweden.The human colorectal cell line Colo205, the human B cell lymphoma cellline Raji and the murine B16 melanoma cell line were from the AmericanTissue Culture Collection (ATCC, Rockville, Md.). The mouse melanomaB16-C215⁺ cells transfected with the expression vector pKGE839containing the Ep-CAM-1 gene (C215) has been described previously (9).

The human cells were cultured in RPMI 1640 medium (Gibco, Middlesex, UK)supplemented with 10% heat inactivated foetal bovine serum (Gibco) and0.1 mg/ml gentamycin sulphate (Biological Industries, Kibbutz BeitHaemek, Israel). The mouse cells were cultured in medium additionallysupplemented with 1 mM glutamine (Hyclone, Cramlington, UK), 5×10⁻⁵ Mβ-mercaptoethanol (ICN, Costa Mesa, Calif.), 0.2% NaHCO₃ (SeromedBiochrome, Berlin, Germany), 1×10⁻² M HEPES (HyClone, Utah) and 1×10⁻³ Msodium pyrovate (HyClone). The cells were repeatedly tested forMycoplasma contamination with Gene-Probe Mycoplasma T. C. test (SanDiego, Calif.).

Phagemid Vector and Phage Library Construction

Total spleen RNA was extracted from one of the responding monkeys usingan RNA isolation kit from Promega (Mannheim, Germany) and cDNA wasamplified using an RNA PCR kit from PE Biosystems (Stockholm, Sweden).The primers for cDNA synthesis of lambda light chain and heavy chaingenes and for the assembly of these genes to scFv genes have beenreported previously (4). The scFv cDNA was ligated into a phagemidvector (4) in fusion with the residues 249-406 of the M13 gene III. ThescFv-gIII gene was expressed from a phoA promoter and the resultingprotein was directed by the E. coli heat stable toxin II signal peptide.

Repeated electroporations of 7 μg library vector with scFv gene insertsresulted in a total of 2.7×10⁷ primary transformed E. coli TG-1 growingas colonies on minimal agar plates. The colonies were scraped from theplates and grown in 2xYT at 150 rpm and 37° C. for 1 h. The culture wassuperinfected with M13K07 helper phage (Promega) in 50 times excess.Ampicillin to a concentration of 100 mg/l was added and the culturegrown for a further hour. After addition of kanamycin to a concentrationof 70 mg/l, the culture was grown for 15 h at 30° C. and 250 rpm. Thephage particles were harvested from the culture supernatant using tworepeated PEG/NaCl precipitations. The precipitated phage was resolved inPBS 1% BSA.

Western Blot Analysis

A two-fold dilution series of scFv-C215 phage particles (from anundiluted stock of PEG-precipitated/concentrated phage) was applied toseparation on a reducing 12% polyacrylamide gel with 1% SDS and 2%β-mercaptoethanol. The proteins were transferred to a nitrocellulosemembrane (Bio-Rad, Hercules, Calif.) by electrophoresis. The membranewas blocked with 5% low-fat milk (Semper AB, Stockholm, Sweden) and thenincubated with a rabbit antiserum against a protein III derived peptidesequence, AEGDDPAKAAFNSLQASATEC, conjugated to keyhole limpethemocyanin. Secondary horse radish peroxidase (HRP) conjugatedgoat-anti-rabbit antibodies (Bio-Rad) were incubated for 30 min. Betweenall steps the membrane was washed 3 times during 5 min in PBS/0.5% Tween20. The membrane was incubated in substrate (Amersham Pharmacia Biotech,Little Chalfon Buckinghamshire, UK) for one min. A light sensitive film(ECL hyperfilm, Amersham) was exposed to the membrane and developed for0.5-5 min.

Similarly, to analyse the integrity of purified Fab (A3, includingcynomolgus CH1 and Clambda domains), scFv- and Fab (including murine CH1and Ckappa)-SEA(D227A) fusion proteins (produced as described previously(9)), 12% SDS-PAGEs were performed. The membranes with transferredproteins were incubated with purified polyclonal rabbit anti-SEAantibodies followed by the reagent steps described above.

Model and Library Phage Selection on Cells

Phage suspensions of the lambda light chain library (or of model phage),10¹² in 100 μl PBS/1% BSA, were incubated with 3 million Colo205 cellsfor 1 h on ice. The cells were washed 3 times including a 10-minincubation using 2 ml PBS/1% BSA for each wash. The phage were eluted byadding 50 μl of 33 μg/ml Genenase to the cell pellet and incubated for15 min. Genenase, which is a subtilisin BPN′ mutant,S24C/H64A/E156S/G169A/Y217L, was kindly provided by Dr. Poul Carter (SanFrancisco, Calif.). After centrifugation the supernatant was transferredto a new tube and 250 μl 1% BSA in PBS was added. To rescue and amplifythe selected library (and the model phage particles in the multi-passexperiment), the eluted phage particles were allowed to infect 1 ml, E.coli DH5αF′ (OD_(600 nm)=1.0). The infected bacterial culture wasdiluted 100 times with 2xYT supplemented with the proper antibiotic andcultured until an OD>1.0 (up to two days).

Finally, to produce soluble scFv the amber suppressor strain HB2151 ofE. coli was infected with the selected library from the second and thirdround. After growth on agar plates containing ampicillin, singlecolonies were cultured in 96 Micro well plates in 2xYT mediumsupplemented with ampicillin at 30° C. for 17 h. After centrifugation,removal of the supernatant to which an equal volume of PBS/1% BSA wasadded, individual scFvs were analysed for immunoreactivity to sectionsof human tumours and normal tissues. Briefly, the C-terminal tag,ATPAKSE, was detected using a rabbit antiserum followed by biotinylatedgoat anti-rabbit antibodies (DAKO A/S, Copenhagen, Denmark) andStreptABComplex HRP (DAKO A/S) (see “Immunohistochemistry”).

Immunohistochemistry

Frozen cryosections (8 μm) were air-dried on slides, fixed in acetone at−20° C. for 10 min and rehydrated in 20% foetal bovine serum in PBS(FBS). Endogenous biotin was blocked with avidin (diluted ⅙) for 15 minand then with biotin (diluted ⅙) for 15 min (Vector Laboratories,Burlingame, Calif.). Affinity purified and biotinylated rabbit anti-SEAantibodies, 5 μg/ml, were incubated for 30 min followed byStreptABComplex HRP (DAKO A/S, Copenhagen, Denmark), 1/110 diluted in 50mM Tris pH 7.6 for 30 min. Between all steps the sections were washed 3times in TBS. The staining reaction was developed for 8 min in 0.5 mg/ml3,3′-diaminobenzidine tetrahydrochloride (Sigma) dissolved in Tris pH7.6 with 0.01 percent H₂O₂. After 10 min counterstaining in 0.5% methylgreen, the slides were rinsed for 10 min in tap water and graduallydehydrated in 70-99% ethanol and xylene before mounting in DPX medium(Sigma).

Flow Cytometry

The Colo205 colon cancer cells were dissociated with 0.02% w/v EDTA andwashed with PBS. To follow the development of an antibody response inthe monkeys the cells were incubated consecutively with diluted serum,for 1 h at 4° C., biotinylated rabbit anti-human IgG antibodies(Southern Biotechnology Ass. Inc., Alabama, USA) for 30 min, and finallywith avidin-PE (Becton Dickinson, Mountain View, Calif.) for 30 min.

The binding of model phage to the cells was analysed usingrabbit-anti-M13 antibodies (produced by immunisation of rabbits with M13particles) and FITC conjugated donkey anti-rabbit antibodies (AmershamPharmacia Biotech). The binding of antibodies fused to SEA(D227A) wasdetected using biotinylated rabbit anti-SEA antibodies and avidin-PE.All reagents were diluted in PBS/1% BSA. The cells were washed twicewith PBS/1% BSA after incubations with reagents and three timesincluding 10 min incubations after binding of phage particles.

Flow cytometric analysis was performed using a FACSort flow cytometer(Becton Dickinson).

Affinity Determination on Cultured Cells

A3 scFv-SEA(D227A), A3 Fab-SEA(D227A) and 1F scFv SEA(D227A) fusionproteins, 80 μg of each protein, were labelled with iodine as describedby Bolton and Hunter to a specific activity of 10-15 μCi/μg. Colo205cells and Raji cells, 30 000/sample were incubated with the iodinatedfusion protein at 100 μl/tube in a two-fold dilution series in 1% BSAfor 1 h and then washed three times in PBS before measuring boundactivity. The concentration of added and bound fusion protein was usedfor Scatchard analysis. The background binding to the Raji cells wassubtracted to calculate the specific binding to the Colo205 cells.

Cytotoxicity Assay

The T cell dependent cytotoxicity of the super-antigen fusion protein(superantigen antibody dependent cellular cytotoxicity, SADCC) wasmeasured in a standard 4 h chromium-release assay employing⁵¹Cr-labelled Colo205 cells as target cells and human T cells aseffector cells (9). The percent specific lysis was calculated as:$100 \times \frac{\begin{matrix}{{{cpm}\quad{experimental}\quad{release}} -} \\{{cpm}\quad{background}\quad{release}}\end{matrix}}{\begin{matrix}{{{cpm}\quad{total}\quad{release}} -} \\{{cpm}\quad{background}\quad{release}}\end{matrix}}$

EXAMPLE 1

Generation of Tumour Binding Monoclonal Cynomolgus Antibodies

Cynomolgus monkeys, Macaca fascicularis (four individuals) wererepeatedly immunised with a suspension of human colon carcinomas fourtimes every other week. The gradual development of an antibody responsein the monkeys was followed by flow cytometric staining of culturedcolorectal cells, Colo205, using dilution series of the preimmune andimmune sera. An IgG antibody response was elicited only when alumprecipitated tumour tissue suspensions were used (two individuals).

The monkey with the highest binding level of immune to preimmune serumantibodies was used for the construction of a large combinatorial scFvphage library of approximately 2.7×10⁷ (estimated from the number ofprimary transformants). The primate phage library was selected usingColo205 cells. The total phage yield (eluted/added number of phagecounted as colony forming units, CFU) from three consecutive selectionrounds increased gradually from 1.9×10⁻⁷, 1.4×10⁻⁵, to 1.2×10⁻³. Fivepercent (12/246) of the monoclonal soluble scFv:s produced from thephage library after the third round of selection were demonstrated tobind to sections of a human colorectal cancer tissue and to intactColo205 cells by flow cytometry. All of the selected antibodiesdemonstrated individually unique nucleic acid sequences according toHinf I restriction patterns analysed by 1% agarose gel electrophoresis.

The antibody genes were amplified by polymerase chain reaction using 5μl of bacterial cultures and primers complementary to regions 5′ and 3′to the scFv gene in the phagemid vector (regions in the phoA promoterand in the M13 gene III).

The Selected scFv Demonstrate Individually Unique Reactivity withEpithelia in Normal Tissues

The colorectal cancer reactive scFv's were classified into specificitygroups based on their immunohistochemical reactivity pattern with normaltissues (Table 1). The antibodies studied in detail were A3 scFv (and A3scFv-SEA(D227A)), A10 scFv, 3D scFv and 1D scFv. The representativeantibodies could be distinguished from each other by their finespecificity to epithelia in different organs and by their binding toleukocytes. The 1D scFv strongly reacted with gut epithelia and was theonly antibody that reacted with cells of polymorph nuclear granulocytemorphology. The 1D scFv also differed from the other antibodies bystaining the luminal surface of kidney tubuli and collecting ductswhereas the A10 scFv reacted homogeneously (non-polarly) with theseepithelial cells and 3D scFv and A3 scFv were negative. 1D, A10 and 3D,but not A3 scFv also reacted with macrophage-like cells in the lung.

A fifth group of antibodies, not extensively evaluated and thus notincluded in Table 1, reacted with colon epithelia, leukocytes andKuppfer cells in the liver. The A3 scFv stands out as demonstrating themost restricted reactivity with the panel of normal tissues used. Themost prominent normal tissue reactivity of the A3 was staining of normalcolon epithelium. Weak staining were also detected in small ducts of thepancreas and bile ducts of the liver and of substructures in small bowelepithelia. The surface epithelium of one of the two stomach samples wasstrongly stained by the A3 antibody.

The reactivity pattern of the A3 scFv was confirmed using the fusionprotein A3 scFv-SEA(D227A). This format permitted the use of polyclonalrabbit anti-SEA antibodies for immunohistochemical detection, which is amore sensitive detection system demonstrating lower background andtissue crossreactivity as compared to the use of secondary antibodies tothe peptide tag, ATPAKSE, at the C-terminus of the scFvs. TABLE 1Immunohistochemical reactivity to normal human tissues of soluble scFvfragments from the selected colorectal cancer phage library scFv clonedesignation Tissue/sub-structure n* A3** A10 3 D 1 D Esophagus/epithelial tissue 1 0 ND ND ND /non-epithelial tissue 0 ND ND ND Colon/epithelium 5 ++ + + ++ /non-epithelial tissue 0 0 0 granulocytes ++Small bowel /villi epithelium 2 (+) heterogenously + + heterogenously(+) /basal glandulae + + + ++ /non-epithelial tissue 0 0 0 0 Ventricle/surface epithelium 2 0, ++ 0 0, + ++ /glandular epithelium 0 +, ++ 0 ++/non-epithelial tissue 0 0 0 0 Pancreas /acini 1 0 (+) + ++ /small ducts(+) (+) + ++ /large ducts 0 (+) + ++ /non-epithelial tissue 0 0 0 0/endocrine 0 0 0 0 Liver /hepatocytes 2 0 ND ND ND /Kuppfer cells 0 NDND ND /bile ducts (+) ND ND ND Kidney /proximal tubuli 1 0 + 0 luminalsurface ++ /distal tubuli 0 + 0 luminal surface ++ /collecting ducts 0 +0 luminal surface ++ /glomeruli 0 0 0 0 /non-epithelial tissue 0 0 0 0Bladder /epithelial tissue 1 0 ND ND ND /non-epithelial tissue 0 ND NDND Prostate /epithelial tissue 1 0 ++ + and secreted material ++/non-epithelial tissue 0 0 0 0 Lung /bronchial epithelium 1 0 (+) (+) 0/alveolar epithelium 0 (+) (+) 0 /non-epithelial tissue 0 macrophages +macrophages + granulocytes ++, macrophages + CNS /gray matter 1 0 ND NDND /white matter 0 ND ND ND Skeletal muscle 1 0 ND ND NDNotes to Table 10 = negative,(+) = weak,+ = moderate,++ = strong,ND = not determined*Number of tissue samples examined**The reactivity of A3 scFv has been confirmed with the A3 scFvSEA(D227A) fusion protein

EXAMPLE 2

The A3 Tumour-Associated Antigen is Homogeneously and FrequentlyExpressed in Colorectal and Pancreatic Tumours

The A3 scFv-SEA(D227A) fusion protein was used for immunohistochemicalstaining of various tumours of epithelial origin (Table 2 and FIG. 1).The fusion protein homogeneously and strongly stained 11/11 samples ofprimary colon cancer tissues and 4/4 metastatic colon cancer samplesresected from the ovary, a lymph node and the liver. Pancreatic cancertumours, 4/4 samples, were equally strongly positive. In contrast,tissue samples of gastric, prostate, breast and non-small cell lungcarcinomas were negative. TABLE 2 Tumor tissue reactivity of A3 scFvSEA(D227A) Tumor tissue n Reactivity Colon cancer, 11 All tumor cellsare strongly and primary tumors homogenously stained Colon cancer 4 Asabove metastasis in lymph node, liver and ovary Pancreas cancer 4 Asabove Ventricle cancer 2 Negative Prostate cancer 2 Negative Breastcancer 2 Negative Lung carcinoma 2 Negative (non-small cell) Malignant 2Negative melanoma

EXAMPLE 3

The A3 TAA is Highly Expressed on the Surface of Colon Cancer Cells

The results from several Scatchard plots for affinity determination,based on the binding of the fusion proteins A3 scFv-SEA(D227A), A3 Faband 1F scFv-SEA(D227A) (1F was classified to the A3 specificity group)to Colo205 cells, are summarised in Table 3. Specific binding wascalculated by subtraction of non-specific binding to human B celllymphoma Raji cells, a cell line not expressing the A3 and 1F TAAs, fromthe binding to Colo205 cells. Linear regression was used to calculatethe slope and the intercept of the extrapolated line in the Scatchardplot. The A3 scFv-SEA(D227A) fusion protein saturated approximately10-fold less binding sites per cell as compared to the A3 Fab (approx. 3million sites per cell) fusion protein, indicating that divalent(multivalent) binding was involved for the scFv. This is supported bythe more than 100-fold higher overall affinity (3.6-5.5 nM) for the A3scFv fusion protein as compared to the A3 Fab (580-780 nM).

A single experiment performed using the 1F scFv-SEA(D227A) fusionprotein, demonstrated similar binding affinity and saturation of bindingsites as the A3 scFv-SEA(D227A) fusion protein. TABLE 3 Scatchardanalysis of iodinated fusion proteins binding to Colo205 cells Fusionprotein n* Kd (nM) million sites/cell A3 Fab-SEA(D227A) 2 580-7803.0-3.9 A3 scFv-SEA(D227A) 3 3.6-5.5 0.11-0.39 1F scFv-SEA(D227A) 1 4.20.18*Number of experiments performed

EXAMPLE 4

A3 and 1F scFv-SEA(D227A) Mediate T Cell Lysis of Colo205 Cells

The capacity of the two fusion proteins A3 and 1F scFv-SEA(D227A) tomediate superantigen antibody dependent cellular cytotoxicity (SADCC)towards Colo205 cells was investigated and compared with the positivecontrol C215 Fab-SEA(D227A) and negative control D1.3 scFv-SEA(D227A)fusion proteins. The A3 scFv-SEA(D227A) fusion protein titrationapproached a plateau for maximal lysis which was similar, approx. 50percent in this 4 h assay, to that demonstrated for the C215Fab-SEA(D227A) fusion protein, although at a ten-fold higherconcentration (FIG. 2). The 1F scFv-SEA(D227A) mediated a similar levelof cytotoxicity at a slightly higher concentration as compared to the A3scFv-SEA(D227A).

The negative control D1.3 scFv SEA(D227A) fusion protein did not mediateany cytotoxicity.

EXAMPLE 5

Purification of a Tumour Associated Antigen that is Recognised by theColon Cancer Reactive Antibody A3.

A tumour extract was made out of xenografted tumour cell line Colo205.The extract was applied to a pre-column coupled with C215Fab-SEAm9, anda column coupled with A3scFv-SEAm9. The columns were in series, duringthe application of sample but separated prior to elution under alkalineconditions.

A single peak was detected during elution by UV spectroscopy (FIG. 3).This eluted fraction from the latter A3-column was collected,neutralised, concentrated, and then analysed by SDS-PAGE undernon-reducing conditions (FIG. 4). Two bands visible by silver staining(labelled I and II in FIG. 4) of apparent molecular weight ofapproximately 90-140 kDa were cut out and examined by standard peptidmapping methodologies. These two bands corresponds to bands detected byA3 in Western Blot, see example 8. From band I 47 separate trypticpeptide masses were obtained (see SEQ ID NO: 3, Table 4, and FIG. 5 forthe sequnces and corresponing mass weights) which completely matched todifferent tryptic peptide masses as determined by MALDI-TOF) of thehuman α6 integrin or β4 integrin (see SEQ ID NOs: 5-51 and 3-4,respectively, and FIGS. 3A and B, respectively, where in FIG. 3A theunderlinings correspond to the peptides appearing in FIG. 3B/SEQ ID NOs:5-51). From band II 22 separate tryptic peptide masses were obtainedwhich completely matched to different tryptic peptide masses of β4integrin (data not shown). The data show that the α6β4 integrinheterodimer is specifically isolated with the A3-affinity column. TABLE4 Peptides/polypeptides derived from human α6β4 integrin and massesthereof Sequence Measured Calculated No. Sequence Mass Mass 5 LLLVGAPR838.568 838.551 6 ANRTGGLYSCDITARGPCTR 2226.131 2226.050 7 VVTCAHRYEK1262.637 1262.631 8 RQHVNTK 882.524 882.490 9 CYVLSQNLR 1152.6181152.583 10 FGSCQQGVAATFTK 1501.706 1501.710 11 DFHYIVFGAPGTYNWK1914.881 1914.917 12 DEITFVSGAPR 1191.625 1191.600 13 ANHSGAVVLLK1108.600 1108.647 14 DGWQDIVIGAPQYFDR 1879.865 1879.897 15DGEVGGAVYVYMNQQGR 1842.811 1842.844 16 WNNVKPIR 1026.608 1026.584 17NIGDINQDGYPDIAVGAPYDDL 2520.213 2520.189 GK 18 GISPYFGYSIAGNMDLDR1975.913 1975.922 19 NSYPDVAVGSLSDSVTIFR 2026.992 2027.008 20 SRPVINIQK1054.644 1054.637 21 LRPIPITASVEIQEPSSR 1993.066 1993.108 22VNSLPEVLPILNSDEPK 1863.920 1864.006 23 TAHIDVHFLK 1180.665 1180.647 24FSYLPIQK 995.601 995.556 25 DIALEITVTNSPSNPR 1726.866 1726.897 26SEDEVGSLIEYEFR 1672.764 1672.770 27 VESKGLEKVTCEPQK 1731.866 1731.895 28REITEKQIDDNRK 1644.792 1644.866 29 FSLFAER 869.476 869.452 30YQTLNCSVNVNCVNIR 1954.003 1953.927 31 LNYLDILMR 1150.644 1150.629 32AFIDVTAAAENIR 1390.739 1390.733 33 LPNAGTQVR 955.523 955.532 34VSVPQTDMRPEK 1386.727 1386.705 35 EPWPNSDPPFSFK 1547.730 1547.717 36NVISLTEDVDEFR 1536.744 1536.754 37 TQDYPSVPTLVR 1375.718 1375.722 38RGEVGIYQVQLR 1417.801 1417.791 39 ALEHVDGTHVCQLPEDQK 2075.965 2075.98140 GNIHLKPSFSDGLK 1512.749 1512.817 41 MDAGIICDVCTCELQK 1928.9011928.822 42 YEGQFCEYDNFQCPR 2012.795 2012.790 43 SCVQCQAWGTGEKKGR1879.865 1879.890 44 DEDDDCTYSYTMEGDGAPGPNS 3103.229 3103.278 TVLVHK 45QEVEENLNEVYR 1521.779 1521.718 46 VAPGYYTLTADQDAR 1640.779 1640.791 47VPLFIRPEDDDEK 1572.778 1572.790 48 DVVSFEQPEFSVSR 1625.758 1625.781 49LLELQEVDSLLR 1427.760 1427.810 50 VCAYGAQGEGPYSSLVSCR 2060.883 2060.91651 VLVDNPKNR 1054.644 1054.600Materials and Methods.Solubilisation of Tumour Tissue

Human colon cancer tissue expressing the A3 antigen was provided byhospitals in Sweden and stored frozen at −70° C. in the tissue bank atABR. Frozen colon cancer tissues were sliced with a scalpel andtransferred into a tube containing cold isotonic sucrose buffer (0.25Msucrose, 10 mM KCl, 1.5M MgCl₂, 50 mM Tris-HCl pH 7.4 at 25° C.)containing 1% (v/v) Nonidet P-40 (NP-40) and protease inhibitors(Completet™ Protease Inhibitor Cocktail Tablet, Boehringer Mannheim).Tissue was homogenised with an Ultra-Turrax homogeniser and were left tosolubilise at 0° C. The solubilised preparation was centrifuged at11,000 rpm (Hettich centrifuge Universal 30 RF rotor), to remove celldebris. The supernatant was further centrifuged at 108,000 g at 4° C.(Beckman Ultracentrifuge Ti-60 rotor), and finally filtered through a0.2 μm Minisart plus filter (Sartoriuis AG Gottingen Germany).

Affinity Purification of Tissue Antigens

A3scFv-SEAm9 was coupled to a NHS-activated HiTrap® column (PharmaciaBiotech Uppsala Sweden), according to the manufacturer'srecommendations. The control and pre-column were coupled withC215Fab-SEAm9, and the control, pre-column and column were set up inseries. All columns were washed with pre-wash buffer (20 mM Tris HClpH7.5 at 4° C. containing 0.2% NP 40). The extract was loaded onto thecolumn at 0.1 ml/min, and the flow through was recirculated. The columnswere then washed with start buffer. Bound antigen was eluted in a pHgradient of diethylamine starting at pH 7.5 up to 11.0. 2.5 ml of eluantwas collected and concentrated down to 75 μl. The purification wasperformed at 4° C. using an AKTA FPLC system (Amersham Pharmacia BiotechUppsala Sweden). Eluted protein was analysed by SDS PAGE and silverstaining. Individual bands were excised, digested with trypsin and themasses of the peptide were determined using a MALDI-TOF instrument byProtana A/S (Odense, Denmark). The peptide masses were then compared ina computer search with all tryptic peptide masses for each protein inthe SWISSPROT database, a service provided by Protana A/S (OdenseDenmark).

EXAMPLE 6

A3scFv-SEAm9 Detects a Novel α6β4 Integrin Epitope

Commercial antibodies to human α6 integrin and β4 integrin were comparedto A3 on normal and malignant colon sections. The reactivity, shown inFIG. 6, demonstrates that A3 is restricted to the colon epithelium (FIG.6[i]), and malignant cell in the tumour (FIG. 6[ii]). Commercialantibody NKI-GoH3 to α6 integrin, also reacted with normal colon (FIG.6[iii]) and colon cancer (FIG. 6[iv]). Reaction was seen in epithelialcells of colon and malignant cells (arrows) but also in blood vessels(BV), some stromal components (s) and in muscularis mucosae (mm). Thereaction observed with commercial ASC-3 anti-β4 integrin antibody wassimilar to that noted with anti-α6 antibody but weaker, in both normalcolon (v) and colon cancer (vi).

Materials and Methods

Antibody

A3 scFv was selected from the M fascicularis library. The VH and VLgenes from this were released by restriction enzyme digestion and fusedto the Staphylococcal Enterotoxin AE chimeric mutant (D227A) to generatethe A3scFv-SEAm9. This demonstrated very low levels of non-specificbinding and allowed sensitive detection by secondary antibodies. ASC-3anti-human-β4 integrin antibody and NKI-GoH3 anti-human-α6 integrinantibody were from Becton Dickinson (Copenhagen, Denmark)

Immunohistochemistry

Tumour and normal tissue samples were obtained from the Department ofSurgery Lund Hospital. These were rate-frozen in iso-pentane, which hadbeen pre-cooled in liquid nitrogen. Samples were stored at −70° C. untilsectioned. After cryosectioning the sections were air dried over night,fixed in cold acetone and blocked with avidin/biotin (Vector BurlingameCalif.). Primary antibody was then added to the section for one hour.

The secondary antibodies were incubated for 30 minutes followed bystreptavidin-biotin/HRP (Dakopatts Copenhagen Denmark) for a further 30minutes. Extensive washing was perfromed between all these steps with 50mM Tris pH 7.6, 0.15M NaCl. Diaminobenzidine (DAB) was used as chromogenand the sections were counterstained in 0.5% methyl green. Controlsincluded a non-tissue reactive Fab and SEA-D227A or no primary antibody.All antibodies were used at a final concentration of 5 μg/ml. Resultswere expressed as negative, weak, moderate or strong staining.

EXAMPLE 7

The A3 Tumour Associated Antigen Reacted with α6 and β4 IntegrinAntibodies in a Capture ELISA

Crude tumor extract or A3 antigen purified by A3-affinity chromatography(see example 5) was analysed by a capture ELISA. Commercial antibodiyASC-3 specific for beta 4 integrin were used as capture antibody, towhich different dilutions of crude tumor extract was applied. This wasthen chased with A3scFv-SEAm9. Bound A3scFv-SEAm9 was then detected withanti-SEA-HRP (FIG. 7A). In FIG. 7B the commercial anti-α6 integrinantibody NKI-GoH3 was used to capture different dilutions of theconcentrated A3-affinity purified eluate. In a similar way as in FIG. 7Athe captured proteins were chased with A3scFv-SEAm9 and detected withanti-SEA-HRP. In both experiments a concentration dependent signal wasdetected. These results confirm the specificity of A3 to α6β4 interginheterodimer, which was also shown to be specifically isolated from theA3-affinity column in example 5.

Material and Methods

Commercial antibodies NKI-GoH3 or ASC-3 (Becton Dickinson CopenhagenDenmark) 100 μl, were used to coat the well of an E.I.A./R.I.A.-plate(Costar) in 0.05 M NaHCO3, pH 9.6. The reaction was allowed to continueovernight at 4° C., after which the plates were washed 4 times inDPBS+0.05% Tween 20. Wells were then blocked with 200 μl 3% non-fat milkpowder in DPBS+0.05% Tween 20, for 1-2 h at room temperature (RT) withshaking. Wells were again washed as above and 100 μl antigen extractdiluted in 3% non-fat milk powder in DPBS+0.05% Tween 20, was appliedfor 2 h at RT with shaking. Wells were again washed (4×DPBS+0.05% Tween20) after which 100 μl of the primary antibody diluted in 3% non-fatmilk powder in DPBS+0.05% Tween 20 was incuabted for 2 h at RT withshaking. Wells were washed again as above and 100 μl of the secondaryantibody diluted in 3% non-fat milkpowder in DPBS+0.05% Tween 20 wasadded to each well for 1 h at RT with shaking. Wells were again washedas above and colour developed by the addition of 100 μl peroxidasesubstrate (Sigma Fast OPD Peroxidase Substrate Tablet Set P-9187). Thereaction was allowed to continue for 30 min at RT, in the dark andshaking before the reaction was stopped by the addition of 50 μl 3 MH₂SO₄. The absorbance was read at 490 nm.

EXAMPLE 8

Western Blot Analysis of the A3 Tumour Antigen

A3-affinity purified tumour antigen extracts were separated by SDS-PAGEand transferred to membranes for Western blot analysis. Extracts wereapplied directly or heated to 100° C. for 5 minutes or heated to 100° C.for 5 minutes but in the presence of mercaptoethanol (BME) (FIG. 8). Themembranes were then probed with A3scFv-SEAm9 and anti-SEA-HRP oranti-human-α6 integrin or anti-human-β4 integrin antibodies. The anti-β4integrin antibody did not react with any protein on the membrane (FIG.8[ii]). The anti-human-α6 integrin reacted with a major specie withapparent molecular weight between 90-140 kDa in the A3-affinity purifiedtumour antigen extract (FIG. 8[iii]). The same species was also detectedby A3scFv-SEAm9, which also was detected after heating but was muchweaker under reduced conditions (with BME present) (FIG. 8[i]). Themajor band detected in the 90-140 kDa interval corresponds to the bandsin example 5, that were analysed by peptide mapping and were found tocontain α6 integrin and β4 integrin.

Materials and Methods

ASC-3 anti-human-β4 integrin antibody and NKI-GoH3 anti-human-α6integrin antibody were from Becton Dickinson (Copenhagen, Denmark).Samples were resolved by SDS-PAGE in 0.25M tris-glycine pH 8.9 and 0.1%SDS at 100V through the upper gel, then 170V through the resolving gel.Molecular weight standards (Biorad broad Range, Biorad) were included onall gels. Resolved samples were transferred to nitrocellulose (Biorad)in transfer buffer (10 mM Tris base, 2M glycine, 40% (v/v) methanol) at100V for 1 hour. Membranes were blocked with 5% (w/v) BSA/TBS for atleast 2 hours at 4° C., then incubated with the appropriate antibodydiluted in 5% BSA/TBS/0.2% azide. This reaction was allowed to proceedfor at least 2 hours at RT, after which the membrane was washedextensively in TBST-T. Bound antibody was detected by incubation ofmembranes for 1 hour with HRP conjugated antibody diluted in TSB-Tcontaining 5% milk powder. Membranes were then incubated with enhancedchemiluminescence (ECL) detection reagents (Renaissance® NEN™ LifeScience Products, Boston Mass.) for 1 minute and exposed to film for upto 1 hour.

REFERENCES

1. DeCosse J J, Tsioulias G J, Jacobson J S. Colorectal cancer:detection, treatment, and rehabilitation. CA Cancer J Clin 1994; 44:27-42.

2. Riethmuller G, et al. Monoclonal antibody therapy for resected Dukes'C colorectal cancer: seven-year outcome of a multicenter randomizedtrial. J Clin Oncol 1998; 16: 1788-1794.

3. Kuhn J A, Thomas G. Monoclonal antibodies and colorectal carcinoma: aclinical review of diagnostic applications. Cancer Invest 1994; 12:314-323.

4. Tordsson J, et al. Efficient selection of scFv antibody phage byadsorption to in situ expressed antigens in tissue sections. J ImmunolMethods 1997; 210: 11-23.

5. Aujame L, Geoffroy F, Sodoyer R. High affinity human antibodies byphage display. Hum Antibodies 1997; 8: 155-168.

6. Clark R K, Trainer D L, Bailey D S, Greig R G Immunohistochemicalanalysis of antiserum from rhesus monkeys immunized with human coloncarcinoma. Cancer Res 1989; 49: 3656-3661.

7. Lewis A P, et al. Cloning and sequence analysis of kappa and gammacynomolgus monkey immunoglobulin cDNAs. Dev Comp Immunol 1993; 17:549-560.

8. Brodin T N, et al. Man-made superantigens: Tumor-selective agents forT-cell-based therapy. Adv Drug Deliv Rev 1998; 31: 131-142.

9. Dohlsten M, et al. Monoclonal antibody-superantigen fusion proteins:tumor-specific agents for T-cell-based tumor therapy. Proc Natl Acad SciUSA 1994; 91: 8945-8949.

10. Liu C, et al. Eradication of large colon tumor xenografts bytargeted delivery of maytansinoids. Proc Natl Acad Sci USA 1996; 93:8618-8623.

LEGENDS TO FIGURES

FIG. 1 The A3 Tumour-Associated Antigen is Homogeneously Expressed inPrimary and Metastatic Tumours

Immunohistochemical staining of frozen and acetone fixed sections ofhuman tumour tissues using A3 scFv-SEA(D227A) and C215 Fab-SEA(D227A) at70 nM. The A3 scFv fusion protein reacted strongly and homogeneouslywith both primary colon and pancreatic carcinoma resected from tumourpatients. A representative staining of a primary colon cancer is shownfor C215 Fab-SEA(D227A) in (A) and for A3 scFv-SEA(D227A) in (B).Staining by A3 scFv-SEA(D227A) of a colon cancer liver metastasis isshown in (C) and of a primary pancreatic cancer in (D).

FIG. 2 A3 scFv-SEA(D227A) Coated Colo205 Tumour Cells are EfficientlyKilled by T Cells.

Superantigen antibody dependent cellular cytotoxicity (SADCC) towardsColo205 cells mediated by A3 scFv-SEA(D227A) reached the same maximalcytotoxicity as the anti-Ep-CAM fusion protein C215 Fab-SEA(D227A),although at a ten-fold higher concentration. The absence of cytotoxicitymediated by the D1.3 scFv-SEA(D227A) demonstrates the need of a tumourtargeting antibody moiety in the fusion protein.

FIG. 3

Immunoaffinity chromatography of tumor extract on a A3scFv-SEAm9 coupledcolumn. Protein bound to A3 coupled columns was washed extensively theneluted as described in Materials and Methods in Example 5. The elutedfractions were examined by UV spectroscopy (arrow) and a single peakidentified. The sample was eluted with a pH gradient as indicated by anx.

FIG. 4

A3 antigen preparation was separated on a non-reduced SDS PAGE andsilver-stained. Previous Western analysis had defined a molecular weightrange in which the A3 antigen was believed to reside. The bands evidentwithin this region (Labelled I and II) were excised for peptide mappinganalysis

FIGS. 5A and 5B

Epithelial integrin α6β4: complete primary structure of α6 and variantforms of β4 (precursor) (Tamura et al J Cell Biol 111:1593-1604 (1990)).The matched peptides shown in SEQ ID NOs: 5-51 are underlined in thesequences of human α6 (FIG. 5A) integrin and β4 (precursor) (FIG. 5B)integrin as published.

FIG. 6

Immunohistochemistry of normal and malignant colon using A3scFv andcommercial anti-human α6 and β4 integrin monoclonal antibodies.

FIGS. 7A and 7B

Capture ELISA. In FIG. 7A monoclonal antibody ASC-3 specific for β4integrin was used as capture antibody, to which different dilutions ofcrude tumor extract was applied. In FIG. 7B the anti-α6 integrinmonoclonal anti-body NKI-GoH3 was used to capture different dilutions ofthe concentrated A3-affinity purified eluate. In both FIGS. 7A and 7Bthe captured integrin antigen was then successfully detected withA3scFv-SEAm9.

FIGS. 8A and 8B

Western blot analysis of the eluate from the A3-affinity column. Theprimary antibodies used are (i) and (ii) A3scFv-SEAm9, (iii) ASC-3anti-human-β4 integrin antibody and (iv) NKI-GoH3 anti-human-α6 integrinanti-body. Lane A—the eluate was applied directly, lane B—the eluate washeated to 100° C. for 5 minutes, and lane C—the eluate was heated to100° C. for 5 minutes but in the presence of mercaptoethanol. Positionsof molecular weight standards are indicated.

1-16. (canceled)
 17. A target structure displayed in, or on the surfaceof, tumour cells, said target structure a) having the ability of beingspecifically blocked by and to specifically block a binding structure,wherein the binding structure is an antibody or antigen binding fragmentof an antibody or a fragment thereof, and other binding structures withsimilar binding properties, b) being displayed in, and on the surfaceof, human gastrointestinal epithelial cells, c) having substantialhomology with α6 and/or β4 integrin chains or variants thereof,representing a shared or unique epitope, d) being highly expressed onthe surface of tumour cells, and e) being a target for cytotoxiceffector mechanism; wherein the antibody is an antibody, or a fragmentthereof, having a binding structure for a target structure displayed in,and on the cell surface of, human gastrointestinal epithelial tumourcells, said binding structure comprising the complementarity determiningregion (CDR) sequences in the light chain comprising essentially theamino acids number 23-33 (CDR1), 49-55 (CDR2), 88-98 (CDR3) of the aminoacid sequence shown in SEQ ID NO:2, and the CDR sequences in the heavychain comprising essentially the amino acids number 158-162 (CDR1),177-193 (CDR2), 226-238 (CDR3) of the amino acid sequence shown in SEQID NO:2.
 18. A target structure according to claim 17, wherein theantibody or antigen binding fragment is labeled and the binding thereofis inhibited by an unlabeled form of said binding structure and not byother binding structures, and not inhibiting the binding of otherbinding structures having other binding specificities.
 19. A targetstructure according to claim 17, wherein the antibody or antigen bindingfragment comprises one or more of the complementarity determining region(CDR) sequences comprising essentially the amino acids number 23-33,49-55,88-98,158-162,177-193,226-238 of the amino acid sequence shown inSEQ ID NO : 2, or other binding structures with similar unique bindingproperties.
 20. A target structure according to claim 17, wherein theantibody or antigen binding fragment is an antibody.
 21. (canceled) 22.A target structure according to claim 17, which is expressedhomogenously in human colonic epithelial cells and less in pancreaticduct and bile duct cells.
 23. A target structure according to claim 17,the expression of which is correlated to gastrointestinal epithelialdifferentiation.
 24. A target structure according to claim 17, whichcomprises essentially the amino acid sequence of a6 integrin shown inSEQ ID NO: 3 and/or of β4 integrin shown in SEQ ID NO: 4, and/or one ormore fragments, and/or variants or splice variants, and or subunits,thereof.
 25. A target structure according to claim 24, which compriseshomo- or hetero-monomers or homo- or hetero-multimers of said α6p4integrin and/or of said one or more fragments and/or variants and/orsubunits thereof.
 26. A target structure according to claim 24, whichhas an apparent molecular weight in its non-reduced form of from 90 to140 kDa, most preferred from 80 to 160 kDa.
 27. A target structureaccording to claim 24, which comprises a peptide or polypeptide(s)comprising essentially any one of the amino acid sequences shown in SEQID NOs: 5-51, or comprises a molecule complexed to said polypeptide(s).28. A target structure according to claim 24 recognised, exclusively ornot, in its non-reduced form by the antibody or antigen binding fragmentcomprised by an antibody or a fragment thereof, having a bindingstructure for a target structure displayed in, and on the cell surfaceof, human gastrointestinal epithelial tumour cells, said bindingstructure comprising the complementarity determining region (CDR)sequences in the light chain comprising essentially the amino acidsnumber 23-33 (CDR1), 49-55 (CDR2), 88-98 (CDR3) of the amino acidsequence shown in SEQ ID NO:2, and the CDR sequences in the heavy chaincomprising essentially the amino acids number 158-162 (CDR1), 177-193(CDR2), 226-238 (CDR3) of the amino acid sequence shown in SEQ ID NO:2.29-34. (canceled)
 35. A pharmaceutical composition comprising as anactive principle a target structure as defined in claim
 17. 36-52.(canceled)
 53. A method for therapy of human malignant disease, wherebyan antibody or a fragment thereof is administered to a human subject,whereby said antibody or fragment thereof has been changed by beinggenetically linked to molecules giving the combined molecule changedpharmaco-kinetic properties; wherein the antibody is an antibody or afragment thereof, having a binding structure for a target structuredisplayed in and on the cell surface of, human gastrointestinalepithelial tumor cells, said binding structure comprising thecomplementary determining region (CDR) sequences in the light chaincomprising essentially the amino acids number 23-33 (CDR1), 49-55(CDR2), 88-98 (CDR3) of the amino acid sequence shown in SEQ ID NO: 2,and the CDR sequence in the heavy chain comprising essentially the aminoacids number 158-162 (CDR1), 177-193 (CDR2), 226-238 (CDR3) of the aminoacid sequence shown in SEQ ID NO:
 2. 54. (canceled)